Method for Producing Basic Substance

ABSTRACT

A method for producing a basic substance by fermentation comprising culturing a microorganism having an ability to produce the basic substance in a liquid medium contained in a fermentation tank to produce and accumulate the basic substance in the medium, wherein amount of sulfate and/or chloride ions used as counter ions of the basic substance is reduced by adjusting total ammonia concentration in the medium to be within a specific concentration range during at least a part of the total period of culture process.

This application is a continuation of, and claims priority under 35U.S.C. §120 of U.S. patent application Ser. No. 13/404,131, filed Feb.24, 2012, which was a divisional under 35 U.S.C. §120 of U.S. patentapplication Ser. No. 12/834,381, filed Jul. 12, 2010, now U.S. Pat. No.8,198,053, issued Jun. 12, 2012, which was a divisional under 35 U.S.C.§120 of Ser. No. 11/697,794, filed Apr. 9, 2007, now U.S. Pat. No.7,790,422, issued Sep. 7, 2010, which was a continuation under 35 U.S.C.§120 of PCT/JP2005/018657, filed Oct. 7, 2005, and claimed priorityunder 35 U.S.C. §119 to JP2004-295123, filed Oct. 7, 2004, all of whichare incorporated by reference. The Sequence Listing filed electronicallyherewith is also hereby incorporated by reference in its entirety (FileName: 2014-09-11T_US-328C4_Seq_List; File Size: 55 KB; Date Created:Sep. 11, 2014).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a technique for the microbial industry,more precisely, a method for producing a basic substance byfermentation. As basic substances which are able to be produced byfermentation, for example, L-lysine is useful as an additive in animalfeed, and L-arginine and L-histidine are useful for pharmaceuticalpreparations such as infusions.

2. Brief Description of the Related Art

In the methods for producing basic substances by fermentation,microorganisms having an ability to produce a basic substance arecultured to produce and accumulate the basic substance in a medium, andthe basic substance is collected from the medium. In such methods, theculture is performed as batch culture, feeding culture or continuousculture.

In such production of basic substances by fermentation, sulfate orchloride ions have been typically added to a medium as counter anionsfor an objective substance which dissociates into a cation in the mediumin order to maintain pH of the medium at a neutral level (JapanesePatent Laid-open (Kokai) Nos. 5-30985 and 5-244969).

In many cases, basic substances are collected from a medium by ionexchange, when purification is required. For example, in the case ofL-lysine, after fermentation broth is made weakly acidic, L-lysine isadsorbed on an ion exchange resin and then eluted from the resin withammonium ions. The eluted L-lysine is used as it is as lysine base, orit can be crystallized with hydrochloric acid to form L-lysinehydrochloride.

When chloride ions are used as counter anions in the medium in theaforementioned purification of L-lysine, L-lysine hydrochloride can beobtained directly by concentrating the medium. However, since chlorideions corrode metal fermentation tanks etc., it is not preferable to makethem exist in the medium in high concentration in actual production.

On the other hand, when the basic substance is not purified, thefermentation broth is concentrated as it is, or it is made weakly acidicwith hydrochloric acid or sulfuric acid, followed by spray granulation.In this case, the residual components contain the counter anions addedto the medium, and therefore the amount of the basic substance isreduced in the resulting fermentation product.

Japanese Patent Laid-open No. 2002-65287 (U.S. Patent Application No.2002025564) discloses a method of utilizing, in the production of abasic amino acid by fermentation, carbonate and bicarbonate ions ascounter anions of the basic amino acid to substitute for a part ofsulfate or chloride ions. Carbonate and bicarbonate ions can becomparatively easily removed from the culture medium by making the pH ofthe medium acidic, or concentrating the medium, or both. The above-citedpublication teaches a method of controlling the internal pressure in thefermentation tank so that it is positive during fermentation, or addingcarbon dioxide gas or a mixed gas containing carbon dioxide to themedium, as a means for adding carbonate ions and bicarbonate ions to themedium. However, at typical medium conditions, such as a neutral pH,only a small amount of carbon dioxide gas dissolves, if at all.Therefore, to maintain the presence of bicarbonate and carbonate ions inthe culture medium so that the effect of reducing the sulfate orchloride ion concentration is maintained, the culture must be performedat an alkaline pH. However, if pH becomes high, the bacterial growthrate and productivity of the objective substance are generally reduced.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for achievingboth reduction of sulfate ions and chloride ions and efficientproduction of an objective substance in the production of a basicsubstance by fermentation using a microorganism having an ability toproduce the objective basic substance and utilizing carbonate ions andbicarbonate ions as counter anions of the basic substance with avoidingreduction of growth rate of the microorganism or reduction ofproductivity of the objective substance.

When using coryneform or Escherichia bacteria to produce basicsubstances by fermentation, if pH becomes too high, the bacterial growthrate or the productivity of the objective substance is usually reduced.The inventors of the present invention found that the major factorcausing this phenomenon is ammonia, which is added to the medium as anitrogen source for production of the basic substance, for bacterialgrowth, or as a source of counter ions of the basic substance, andreduction of the growth rate of the microorganism or the productivity ofthe objective substance under a high pH condition could be markedlysuppressed by performing the fermentation with controlling the totalammonia concentration to be within a suitable concentration range.

The present invention was accomplished on the basis of theaforementioned findings.

That is, the present invention provides the followings.

(1) A method for producing a basic substance by fermentation comprisingculturing a microorganism having an ability to produce the basicsubstance in a liquid medium contained in a fermentation tank to produceand accumulate the basic substance in the medium, wherein amount ofsulfate and/or chloride ions used as counter ions of the basic substanceis reduced by adjusting total ammonia concentration in the medium to bewithin a specific concentration range during at least a part of thetotal period of culture process.

(2) The method according to (1), wherein the specific concentrationrange of the total ammonia concentration is a range satisfying thefollowing conditions:

(A) concentration of ammonium ions in the medium is at such a level thatthe sum of the ion equivalents of bicarbonate ions and/or carbonate ionsand other anions dissolved in the medium is larger than the ionequivalent of the basic substance ionized from the basic substanceaccumulated in the medium, and

(B) the total ammonia concentration in the medium is at a level notinhibiting the production of the basic substance by the microorganism,which is determined beforehand as follows:

the microorganism is cultured in the medium having various pH values andvarious total ammonia concentrations, productivity of the basicsubstance is measured at each pH value and each total ammoniaconcentration, and a total ammonia concentration providing 50% or moreof productivity of the basic substance based on the productivityobtained under optimum conditions is determined for each pH value.

(3) The method according to (1), wherein the specific range of the totalammonia concentration is determined beforehand as follows:

(A′) the culture is performed in a medium which contains sulfate and/orchloride ions in an amount sufficient for performing the culture at pH7.2 or lower as a source of counter ions of the objective basicsubstance, of which pH is maintained to be in the range between 6.5 to7.2 by adding at least one of ammonia gas, aqueous ammonia and urea, andproductivity of the basic substance is measured,

(B′) the culture is started in the same medium as that used in the step(A′) except that sulfate ions and/or chloride ions of medium componentsare lowered by a desired amount, and the culture is continued withvarious total ammonia concentrations during a period where it becomesimpossible to maintain the pH of the medium to be 7.2 or lower due tothe shortage of sulfate ions and/or chloride ions as counter ions of thebasic substance caused by accumulation of the objective basic substanceto determine a total ammonia concentration range providing 50% or moreof productivity based on the productivity measured in the step (A′).

(4) The method according to (1), wherein the at least a part of thetotal period includes at least one of a period where the pH of themedium increases due to shortage of the counter ions caused withaccumulation of the objective basic substance, and a period where the pHincreases due to the addition of cations to the medium.

(5) The method according to (1), wherein the total ammonia concentrationin the medium is adjusted by adding ammonia or urea to the medium whenthe activity of the microorganism is reduced or ceases as determinedbased on the indicators: dissolved oxygen concentration in the medium,consumption rate of carbon source in the medium, turbidity of themedium, productivity of the basic substance, and pH change in the mediumobserved.

(6) The method according to (1), wherein a medium having the samecomposition as that of a medium containing sulfate ions and/or chlorideions as a counter ion source of the basic substance in an amountsufficient for performing the culture at pH 7.2 or lower except thatamount of sulfate ions and/or chloride ions is reduced by a desiredamount is used as the medium, and

the at least a part of the total period is a period where pH of themedium cannot be maintained to be 7.2 or lower due to shortage ofcounter ions for the basic substance which has accumulated in themedium.

(7) The method according to (2), wherein the other anions are selectedfrom sulfate ions, chloride ions, phosphate ions, and ionized organicacids.

(8) The method according to (2) or (7), wherein total amount of theother anions is 900 meq/l or lower.

(9) The method according to (1), wherein the total ammonia concentrationin the medium is adjusted to be 200 mM or lower.

(10) The method according to (1), which comprises the step ofproliferating the microorganism.

(11) The method according to (10), wherein the total ammoniaconcentration is not adjusted during the step of proliferating themicroorganism.

(12) The method according to (1), wherein the basic substance isselected from L-lysine, L-arginine and L-histidine.

(13) The method according to (12), wherein the basic substance isL-lysine.

(14) The method according to (12), wherein the basic substance isL-arginine.

(15) The method according to (1), wherein the medium or a processedproduct thereof is heated after the fermentation to eliminatebicarbonate ions and carbonate ions.

(16) The method according to (1), wherein the microorganism is acoryneform bacterium or an Escherichia bacterium.

(17) A fermentation broth or fermentation product containing a basicsubstance, which is obtainable by the method according to (15).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of culture for L-lysine production performed byusing a conventional medium and the culture method. FIG. 1 a shows thegrowth curve over time at two different pH values. FIG. 1 b shows thelysine accumulation over time at two different pH values. FIG. 1 c showsthe ammonia concentration over time at two different pH values.

FIG. 2 shows the results of culture for L-lysine production performed ina medium with a limited ammonium concentration. FIG. 2 a shows thegrowth curve over time at three different pH values. FIG. 2 b shows thelysine accumulation over time at three different pH values. FIG. 2 cshows the ammonia concentration over time at three different pH values.

FIG. 3 shows the results of culture for L-lysine production performed bycontrolling only the total ammonia concentration and not controlling pH.FIG. 3 a shows the growth curve over time. FIG. 3 b shows the lysineaccumulation over time. FIG. 3 c shows the ammonia concentration overtime.

FIG. 4 shows changes in total ammonia concentration (4 a) and pH (4 b)over time in a conventional medium and a medium without ammonium sulfateand ammonium chloride.

FIG. 5 shows the changes in growth (5 a), total ammonia concentration (5b), pH (5 c), and remaining sugar amount (5 d) over time in L-argininefermentation in a medium with a limited ammonium concentration.

FIG. 6 shows the results of a culture for L-arginine production in amedium with a limited ammonium concentration.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereafter, the present invention will be explained in detail.

The method of the present invention is a method for producing a basicsubstance by fermentation, which comprises culturing a microorganismwhich is able to produce the basic substance in a liquid mediumcontained in a fermentation tank to produce and accumulate the basicsubstance in the medium. The method of the present invention ischaracterized in that amount of sulfate ions and/or chloride ions usedas counter ions of the basic substance is reduced by adjusting totalammonia concentration in the medium to be within a specificconcentration range during at least a part of the total period ofculture process. That is, the method of the present invention is amethod for producing the basic substance in the medium in which sulfateions and chloride ions are reduced by using such a total ammoniaconcentration that the total ammonia is secured in an amount requiredfor the growth of the microorganism or the production of the objectivesubstance as a nitrogen source, and growth of the microorganism or theproduction of the objective substance is not inhibited.

Examples of the specific range of the total ammonia concentrationinclude a range satisfying the following conditions:

(A) the concentration of ammonium ions in the medium is at such a levelthat the sum of the ion equivalents of bicarbonate and/or carbonateions, and other anions dissolved in the medium is larger than the ionequivalent of the basic substance ionized from the basic substanceaccumulated in the medium, and

(B) the total ammonia concentration in the medium is at a level notinhibiting the production of the basic substance by the microorganism,which is determined beforehand as follows:

the microorganism is cultured in the medium having various pH values andvarious total ammonia concentrations, productivity of the basicsubstance is measured at each pH value and each total ammoniaconcentration, and a total ammonia concentration providing 50% or moreof productivity of the basic substance based on the productivityobtained under optimum conditions is determined for each pH value.

Furthermore, in another embodiment of the present invention, thespecific range of the total ammonia concentration is determinedbeforehand as follows.

(A′) (Procedure 1: Evaluation of Fermentation Result Under NeutralCondition)

The culture is performed in a medium which contains an amount of sulfateand/or chloride ions which is sufficient for performing the culture atpH 7.2 or lower as a source of counter ions of the objective basicsubstance, of which pH is maintained to be in the range between 6.5 to7.2 by adding at least one of ammonia gas, aqueous ammonia and urea, andproductivity of the basic substance is measured,

(B′) (Procedure 2: Evaluation of Fermentation Results with ReducedAmount of Sulfate Ions and Chloride Ions at Various AmmoniumConcentrations)

The culture is started in the same medium as that used in Procedure 1(step A′) described above except that sulfate and/or chloride ions ofmedium components are lower by a desired amount, and the culture iscontinued with various total ammonia concentrations during a periodwhere it becomes impossible to maintain the pH of the medium to be 7.2or lower due to shortage of sulfate ions and/or chloride ions as counterions of the basic substance caused with accumulation of the objectivebasic substance to determine a total ammonia concentration rangeproviding 50% or more of productivity based on the productivity measuredin the step (A′).

Moreover, in another embodiment of the present invention, when thespecific range of total ammonia concentration is not determinedbeforehand, the total ammonia concentration can be adjusted to be withinthe predetermined range. Specifically, the total ammonia concentrationin the medium is adjusted by adding ammonia or urea to the medium whenactivity of the microorganism is reduced or ceases as determined on thebasis of dissolved oxygen concentration in the medium, consumption rateof carbon source in the medium, turbidity of the medium, productivity ofthe basic substance, and the pH change in the medium observed asindexes. The medium has the same composition as that of a mediumcontaining sulfate ions and/or chloride ions as a counter ion source ofthe basic substance in an amount sufficient for performing the cultureat pH 7.2 or lower except that amount of sulfate ions and/or chlorideions is reduced by a desired amount. Examples of the at least a part ofthe total period include a period when the pH of the medium cannot bemaintained to be 7.2 or lower due to shortage of counter ions for thebasic substance which has accumulated in the medium.

Examples of the other anions include chloride ions, sulfate ions,phosphate ions, ions of organic acids (acetic acid, lactic acid,succinic acid etc.), and so forth. Furthermore, bicarbonate ions and/orcarbonate ions dissolved in the medium function as counter anions of thebasic substance.

In the present invention, an ion equivalent is a value obtained bymultiplying the molar concentration of each ion by the ion's valence,and it is represented in a unit of eq/l. That is, the ion equivalent of1 mM of a monovalent ion is 1 meq/l, and the ion equivalent of 1 mM of adivalent ion is 2 meq/l.

The aforementioned total ammonia concentration is adjusted in order tomake the total ammonia exist in the medium in an amount required forgrowth of the microorganism or the production of the basic substance,and at a concentration not inhibiting the production of the basicsubstance by the microorganism, and the medium is thereby automaticallyadjusted to a pH suitable for dissolving bicarbonate ions and/orcarbonate ions required as counter anions of the basic substance.

In the present invention, “the total ammonia” means the sum ofnon-dissociated ammonia (NH₃) and ammonium ions (NH₄ ⁺). When adjustingthe total ammonia concentration, non-dissociated ammonia or ammoniumions may be measured, or the both may be measured.

Typically, ammonium sulfate and ammonium chloride are added to themedium as sources of counter anions of the basic substance and source ofnitrogen, in general. Moreover, since ammonia and urea are typicallyused to adjust the pH of the medium, high concentration of ammonia andammonium ions are present in the medium. When reducing the amount ofammonium sulfate or ammonium chloride in order to reduce the amount ofsulfate or chloride ions added to the medium, a nitrogen source such asammonia is supplied in an amount corresponding to the amount to bereduced. For such an operation, it has been necessary to develop amethod for supplying ammonia, which takes into consideration the balancebetween cations including those produced by bacteria and increasing withprogress of the culture such as those of the objective basic substance,cations which ionize from added ammonia, cations added to the mediumsuch as sodium and potassium ions, and so forth, and anions increasingin the medium due to generation by respiration of bacteria or additionto the medium. If this balance is not maintained, the fermentation willnot progress, because the ammonia concentration will become unduly high,or the pH will become excessively high, or conversely, ammonia couldbecome depleted. According to the present invention, development of amethod for adding ammonia for adjusting the total ammonia concentrationto be within a specific range can enable favorable maintenance of theaforementioned balance of cations and anions, and thus favorable growthof a microorganism and favorable generation of a basic substance can berealized even under a condition that the amount of sulfate ions andchloride ions present in the medium is reduced.

The total ammonia concentration in the medium is adjusted by adding atleast one of ammonia gas, ammonia solution and urea to the medium sothat the total ammonia concentration in the medium is at an acceptablelevel. Furthermore, an ammonium salt such as ammonium chloride orammonium sulfate may also be added, unless detrimental to the effect ofthe invention. Moreover, an ammonium salt containing bicarbonate ion orcarbonate ion as a counter ion, which can be easily removed as a gasafter completion of the culture, may also be used. The total ammoniaconcentration can be adjusted by using measured values of ammonium ionor ammonia concentration in the medium or exhaust gas as an index.Moreover, it is also possible to adjust the total ammonia concentrationby determining beforehand a pH providing an acceptable total ammoniaconcentration when pH is adjusted with ammonia and adding ammonia sothat such a pH can be obtained. In such a case, pH determined asdescribed above may be changed during the culture, if needed.

Moreover, the total ammonia concentration in the medium can also beadjusted by adding ammonia or urea to the medium when the activity ofthe microorganism is reduced or ceases as determined on the basis ofdissolved oxygen concentration in the medium, consumption rate of carbonsource in the medium, turbidity of the medium, productivity of the basicsubstance, and the change in pH in the medium observed as indexes. Thatis, if the nitrogen source in the medium runs short or is depleted,proliferation of the microorganism or the activity of the microorganism,such as production of an objective substance is reduced or ceases.Activity of a microorganism usually appears as consumption of dissolvedoxygen and a carbon source in a medium, increase of turbidity of medium,production of an objective substance, and reduction in the pH of themedium due to the consumption of ammonia or the release of carbondioxide by respiration. Therefore, when activity of a microorganism isreduced or ceases, the concentration of dissolved oxygen in a mediumincreases when aeration and stirring rates per unit time are constant,and pH of a medium increases due to a decrease in the consumption ofammonia and secretion of carbon dioxide. Furthermore, the consumptionrate of a carbon source, increasing rate of turbidity of a medium andthe production rate of an objective substance are reduced. Therefore,when stagnation of activity of a microorganism is observed on the basisof these items used as indexes under a state that medium componentsother than a nitrogen source are sufficient, the nitrogen source runsshort or has been depleted. If this occurs, ammonia or urea is added tothe medium in an amount which is required for the growth of themicroorganism or the production of the objective substance. By repeatingthis procedure, the total ammonia concentration in the medium ismaintained to be within a specific range as a result. If the culture isperformed with adding urea to the medium, urea is utilized by themicroorganism, and ammonia is released into the medium. If the additionof ammonia or urea is repeated as described above, the pH of the mediumgradually increases. The amount of ammonia or urea added at each timepoint may be, for example, 300 mM, preferably 200 mM, more preferably100 mM, expressed as the final concentration of total ammonia in themedium. Alternatively, ammonia or urea may be added so that the pHincreases by 0.3 or less, preferably 0.15 or less, more preferably 0.1or less, after addition of ammonia or urea.

The dissolved oxygen concentration in the medium can be measured, forexample, by using a dissolved oxygen electrode.

Whether the sum of the ion equivalents of bicarbonate ions and/orcarbonate ions and the other anions, which are all dissolved in themedium is higher than the ion equivalent of the basic substance whichhas accumulated in the medium can be confirmed by measuring theconcentrations of bicarbonate ions, carbonate ions and other anions aswell as the concentration of the basic substance. Moreover, the aboveconditions can also be fulfilled by conducting a preliminary experimentto determine the pH and/or the addition amount of ammonia whichsatisfies the aforementioned conditions, and performing the culture atthe pre-determined pH and/or addition of pre-determined amount ofammonia.

In the present invention, the pH of the culture may or may not beconstant. Moreover, when the pH of the medium is controlled, it may becontrolled by using pH itself as an index, or indirectly by controllingthe total ammonia concentration without directly controlling the pH.Furthermore, if ammonia or urea is added using the activity of themicroorganism as an index as described above, the total ammoniaconcentration in the medium is adjusted so that it is within anappropriate concentration range, and the pH gradually increases with theaccumulation of the basic substance. Moreover, if the culture isperformed with controlling the total ammonia concentration to be withina specific range, the pH changes as a result of change of accumulationbalance of various cations and anions in the medium. Whichever means ischosen, the total ammonia concentration in the medium is adjusted to bewithin a specific concentration range as a result, and thus the amountof sulfate ions and/or chloride ions used as counter ions of the basicsubstance can be reduced.

In the present invention, the expression “not inhibiting production of abasic substance” means that the microorganism used for the presentinvention grows favorably, and the basic substance is favorablyproduced. When the growth of the microorganism is insufficient, or whenthe basic substance is not efficiently produced in spite of favorablegrowth of the microorganism, it is considered that production of thebasic substance is inhibited.

Specifically, the microorganism used for the present invention iscultured at various pH levels and the total ammonia concentrations ofthe medium, productivities of the basic substance accumulated in themedium are measured, and the total ammonia concentrations which resultsin production of the basic substance at a rate of preferably 50% ormore, more preferably 70% or more, particularly preferably 90% or more,as compared to the amount of the basic substance obtainable underoptimal conditions, for example, conventionally used general conditionsat a neutral pH, at each pH value are considered to be concentrations“not inhibiting production of the basic substance”. In the presentinvention, “productivity” refers to the yield, the production rate orthe total amount produced. The “yield” refers to production amount ofthe basic substance based on the carbon source present in the mediumwhich is able to be consumed, and the “production rate” refers to aproduction amount per unit time. Moreover, when the term “productionamount” or “amount produced” is solely used, it refers to the amount ofthe basic substance which is accumulated in the medium once the carbonsource is completely consumed.

Alternatively, the microorganism used for the present invention iscultured under optimal conditions, for example, conventionally usedgeneral conditions at a neutral pH, and productivity of the basicsubstance which has accumulated in the medium is measured. Then, theculture is performed in a medium having the same composition except thatamount of sulfate ions and/or chloride ions is reduced by a desiredamount, and the productivity of the basic substance is measured. In thiscase, there is a period where the pH of the medium will increase due tothe shortage of sulfate ions and/or chloride ions as the counter ionswith accumulation of the objective basic substance. For that period, theculture is performed with maintaining the total ammonia concentration tobe within the specific concentration range. As for the range withinwhich the concentration is controlled, the culture is performed withvarious concentrations within the range of 1 to 500 mM, andconcentrations within a range providing a productivity of the basicsubstance of preferably 50% or more, more preferably 70% or more,particularly preferably 90% or more, of the productivity obtainableunder optimal conditions are determined to be concentrations “notinhibiting production of the basic substance”. Examples of the mediumused for the aforementioned “conventionally used general conditions at aneutral pH” include a medium containing sulfate ions and/or chlorideions in an amount sufficient for performing the culture at pH 7.2 orlower.

The desired amount by which the sulfate ions and/or chloride ions arereduced is not particularly limited, so long as objective productivityof the basic substance can be obtained.

The total ammonia concentration which is defined as “not inhibiting theproduction of the basic substance” can also be determined, for example,as follows. The microorganism used for the present invention is culturedat various pH levels and the total ammonia concentration of the medium,and the amount of the basic substance which accumulates in the mediumare measured. The accumulated amount of the basic substance which isobtained under various conditions are compared with the amountaccumulated under the optimum conditions. Thus, the total ammoniaconcentration which does not inhibit the production of the basicsubstance can be determined. The optimum conditions are defined asconditions of culture using sufficient counter ions at a neutral pH asin the typically used general conditions at a neutral pH.

Furthermore, another method for determining the total ammoniaconcentration which is defined as “not inhibiting production of thebasic substance” is, for example, as follows. The microorganism used forthe present invention is cultured under optimal conditions, for example,typically used general conditions at a neutral pH, and productivity ofthe basic substance which accumulates in the medium is measured. Then,the culture is performed in a medium having the same composition exceptthat sulfate ions and/or chloride ions is reduced by a desired amount,and the productivity is examined. In this case, there is a period wherepH of the medium will increase due to shortage of sulfate ions and/orchloride ions as the counter ions with accumulation of the objectivebasic substance. For that period, the culture is performed withmaintaining the total ammonia concentration to be with in the specificconcentration range. As for the range within which the concentration iscontrolled, the culture is performed with various concentrations withinthe range of 1 to 500 mM, and the productivities obtained thereby arecompared to that under the optimum conditions.

The concentration which is defined as “not inhibiting production of thebasic substance” includes, for example, a concentration allowsproduction of the basic substance preferably at 50% or more, morepreferably 70% or more, particularly preferably 90% or more, as comparedto the productivity of the basic substance under optimal conditions.Specifically, the total ammonia concentration in the medium is, forexample, preferably 300 mM or less, more preferably 200 mM or less,particularly preferably 100 mM or less. The degree to which ammoniadissociates is reduced as the pH increases. Non-dissociated ammonia ismore toxic to bacteria as compared with ammonium ion. Therefore, theupper limit of the total ammonia concentration also depends on the pH ofthe medium. That is, as the pH of the medium increases, the acceptabletotal ammonia concentration becomes lower. Therefore, as for theaforementioned total ammonia concentration which is defined as “notinhibiting production of the basic substance”, a total ammoniaconcentration range acceptable for the highest pH during the culture maybe regarded as the total ammonia concentration range throughout theculture.

On the other hand, the total concentration of ammonia as a nitrogensource, which is required for growth of the microorganism and productionof the basic substance, is not particularly limited, so long as theproductivity of the objective substance provided by the microorganism isnot reduced due to a shortage of the nitrogen source during the culture,and it may be appropriately determined. For example, the ammoniaconcentration is measured over time during the culture, and when theammonia in the medium is depleted, a small amount of ammonia may beadded to the medium. Although the concentration after the addition ofammonia is not particularly limited, it is, for example, preferably 1 mMor higher, more preferably 5 mM or higher, particularly preferably 10 mMor higher, in terms of the total ammonia concentration.

The method of the present invention may include a culture step which isprimarily for proliferating the microorganism having an ability toproduce a basic substance, and a culture step which is primarily forallowing the microorganism to produce the basic substance. Furthermore,in the method of the present invention, proliferation of themicroorganism and production of the basic substance may be performed inparallel. Furthermore, besides such culture as described above, whichmay also be called main fermentation, main culture or the like, apreculture may also be independently performed.

In the present invention, in addition to adjusting the total ammoniaconcentration in the medium as described above, an operationfacilitating dissolution of bicarbonate ions and/or carbonate ions inthe medium may also be performed. Examples of such an operation includecontrolling the pressure in the fermentation tank during the culture sothat it is positive, supplying carbon dioxide gas or a mixed gascontaining carbon dioxide gas to the medium, limiting the aeration inthe fermentation tank so that bicarbonate ions and/or carbonate ions aredissolved in the medium, increasing the pH of the medium by addingcations other than ammonium ions such as sodium ions and potassium ionsto the medium, and so forth.

To make the pressure in the fermentation tank positive, for example, thepressure of the air supply to the fermentation tank may be made higherthan the pressure of the exhaust. By making the pressure in thefermentation tank higher, carbon dioxide gas generated by thefermentation dissolves in the culture medium and produces bicarbonateions or carbonate ions. Specifically, the pressure in the fermentationtank may be 0.13 to 0.3 MPa, preferably 0.15 to 0.25 MPa.

Furthermore, carbon dioxide gas may be dissolved in the culture mediumby supplying carbon dioxide gas or a mixed gas containing carbon dioxidegas into the medium. Alternatively, by limiting aeration to thefermentation tank, carbon dioxide gas generated by the fermentation canalso dissolves in the medium. A suitable aeration rate can be determinedby, for example, measuring the amount of bicarbonate ions or carbonateions in the medium, or measuring the pH and ammonia concentration of themedium. When carbon dioxide gas is supplied to the medium, for example,pure carbon dioxide gas or a mixed gas containing 5% by volume or moreof carbon dioxide gas may be bubbled into the medium. The aforementionedmethods for dissolving bicarbonate ions and/or carbonate ions in themedium may be used independently or as a combination of two or more.

The operation of adjusting the total ammonia concentration in the mediumand the operation of facilitating dissolution of bicarbonate ions and/orcarbonate ions in the medium if needed may be performed during at leasta part of the total period of culture process.

Although the “at least a part of the total period” is not particularlylimited so long as desired productivity is obtained, it may bespecifically, for example, 1/10 or more, preferably ⅕ or more, of thetotal culture process of the main culture. More specifically, examplesof the period include a period where the pH of the medium increases dueto the shortage of the counter ions such as sulfate ions and/or chlorideions, with accumulation of the objective basic substance, or a periodwhere the pH of the medium increases due to addition of cations, or bothof these periods.

The medium used for the present invention is not particularly limited,so long as at least the total ammonia concentration can be made to bewithin the aforementioned range by the operation of adjusting the totalammonia concentration, and a medium containing organic and inorganicnutrients such as a carbon source and a nitrogen source and other traceamount nutrients may be suitably used depending on the microorganism tobe used.

Any carbon source can be used, as long as it can be consumed by themicroorganism, and examples include saccharides such as saccharose,glucose, fructose, molasses and starch hydrolysate, organic acids suchas acetic acid, alcohols such as ethanol, and hydrocarbons such asmethane.

Examples of the nitrogen source include inorganic substances such asammonia, protein hydrolysates, yeast extract, and so forth. Examples ofthe trace amount nutrients include amino acids, vitamins, and tracemetal elements.

Examples of anions other than bicarbonate ions and/or carbonate ionswhich are present in the medium include chloride ions, sulfate ions,phosphate ions, ionized organic acids, hydroxide ions, and so forth. Thesum of the ion equivalents of these other ions is usually 900 meq/l orless, preferably 700 meq/l or less, more preferably 500 meq/l or less,still more preferably 300 meq/l or less, particularly preferably 200meq/l or less.

One of the objects of the present invention is to reduce the amount ofsulfate ions and/or chloride ions used, and the ion equivalent ofsulfate ions or chloride ions, or the sum of ion equivalents of theseions present in the medium is usually 700 meq/l or less, preferably 500meq/l or less, more preferably 300 meq/l or less, still more preferably200 meq/l or less, particularly preferably 100 meq/l or less.

The fermentation scheme is not particularly limited, and may be a batchculture in which medium is not fed, a feeding culture in which themedium is fed after the charged sugar is consumed, a continuous culturein which the medium is extracted when the volume of the medium exceedsthe volume acceptable for a fermentation tank, a cell recycle method inwhich bacterial cells are recycled, and so forth. The culturetemperature may be appropriately determined depending on the chosenmicroorganism. It is usually 25 to 45° C., preferably 30 to 40° C.Furthermore, it is preferable to stir sufficiently so that sufficientoxygen is present during the fermentation.

The culture for producing the objective basic substance is specificallyperformed, for example, as follows. A medium containing typical mediumcomponents is prepared, but most if not all of the ammonium salts suchas ammonium sulfate and ammonium chloride are eliminated. Amicroorganism which has been separately cultured is inoculated into thismedium, and cultured while controlling the total ammonia concentrationto be within a range suitable for the chosen microorganism, which isdetermined as described above. The ammonia concentration in the mediumin the fermentation tank or the sampled medium can be measured by using,for example, a commercially available ion meter or the like. By usingthe measured values as an index, the total ammonia concentration can becontrolled. To maintain the total ammonia concentration within thepredetermined concentration range, ammonia gas, aqueous ammonia or ureamay be added to the medium. The total ammonia concentration in themedium can also be indirectly measured by measuring the ammoniaconcentration in the exhaust gas from the fermentation tank using acommon ammonia electrode.

Furthermore, in the present invention, the total ammonia concentrationin the medium can be adjusted by the following method using the pH ofthe medium as an index, as described above.

The culture is performed in a medium which has the same composition as amedium containing sulfate ions and/or chloride ions in an amountsufficient to maintain the culture at pH 7.2 or lower, except thatamount of sulfate ions and/or chloride ions is reduced by a desiredamount at various pH levels, wherein the pH level is changed by addingat least any one of ammonia gas, aqueous ammonia and urea, and

the culture is continued while maintaining the total ammoniaconcentration in the medium so that it is within the preferredconcentration range by adding at least any one of ammonia gas, aqueousammonia and urea to the medium based on indicators such as change in thedissolved oxygen concentration in the medium, the change in theconsumption rate of the carbon source in the medium, the change in theturbidity of the medium, the pH change in the medium, or the like in anindirect manner during a period where pH of the medium cannot bemaintained at 7.2 or lower due to shortage of counter ions to the basicsubstance which has accumulated in the medium.

Examples of the basic substance produced by the method of the presentinvention include basic amino acids, specifically, L-lysine, L-arginineand L-histidine. Among these, L-lysine is preferred.

The microorganism which is able to produce a basic substance is notparticularly limited, and any microorganism can be chosen so long as itcan produce the basic substance by fermentation. In particular, amicroorganism which favorably produces the basic substance even at ahigh pH of medium, if the total ammonia concentration of the medium islow, is preferably chosen. Examples of such a microorganism includebacteria belonging to coryneform bacteria, genus Escherichia, Serratia,or Bacillus.

Coryneform bacteria and Escherichia bacteria will be explainedhereinafter, however, the microorganism used for the method of thepresent invention is not limited to these bacteria.

The coryneform bacteria used for the present invention includeCorynebacterium bacteria and those bacteria having been previouslyclassified into the genus Brevibacterium but have been re-classifiedinto the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1981)), and further include bacteria belonging to the genusBrevibacterium, which is extremely close to the genus Corynebacterium.Specific examples include the following:

Corynebacterium acetoacidophilum

Corynebacterium acetoglutamicum

Corynebacterium alkanolyticum

Corynebacterium callunae

Corynebacterium glutamicum

Corynebacterium lilium (Corynebacterium glutamicum)

Corynebacterium melassecola

Corynebacterium thermoaminogenes

Corynebacterium efficiens

Corynebacterium herculis

Brevibacterium divaricatum (Corynebacterium glutamicum)

Brevibacterium flavum (Corynebacterium glutamicum)

Brevibacterium immariophilum

Brevibacterium lactofermentum (Corynebacterium glutamicum)

Brevibacterium roseum

Brevibacterium saccharolyticum

Brevibacterium thiogenitalis

Brevibacterium album

Brevibacterium cerinum

Microbacterium ammoniaphilum

Specifically, the following strains are included:

Corynebacterium acetoacidophilum ATCC 13870

Corynebacterium acetoglutamicum ATCC 15806

Corynebacterium alkanolyticum ATCC 21511

Corynebacterium callunae ATCC 15991

Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060

Corynebacterium lilium (Corynebacterium glutamicum) ATCC 15990

Corynebacterium melassecola ATCC 17965

Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)

Corynebacterium herculis ATCC 13868

Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020

Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC14067

Brevibacterium immariophilum ATCC 14068

Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13665,ATCC 13869

Brevibacterium roseum ATCC 13825

Brevibacterium saccharolyticum ATCC 14066

Brevibacterium thiogenitalis ATCC 19240

Brevibacterium ammoniagenes (Corynebacterium ammoniagenes) ATCC 6871

Brevibacterium album ATCC 15111

Brevibacterium cerinum ATCC 15112

Microbacterium ammoniaphilum ATCC 15354

Examples of the Escherichia bacteria include Escherichia coli. WhenEscherichia coli is bred by using genetic engineering techniques, the E.coli K12 strain and derivatives thereof, i.e., E. coli MG1655 strain(ATCC No. 47076), W3110 strain (ATCC No. 27325), and so forth, may bechosen. The E. coli K12 strain was isolated at Stanford University in1922, and is a lysogenic bacterium of λ phage. In addition, it is ahighly versatile strain having the F-factor, for which geneticrecombinants can be created by conjugation or the like. Furthermore, thegenomic sequence of the E. coli K12 strain has been determined, and thegenetic information is publicly available. The E. coli K12 strain andderivatives thereof may be obtained from American Type CultureCollection (ATCC, Address: P.O. Box 1549, Manassas, Va. 20108, UnitedStates of America).

Examples of coryneform bacteria which are able to produce L-lysineinclude S-(2-aminoethyl)cysteine (abbreviated as “AEC” hereinafter)resistant mutant strains, mutant strains which require an amino acidsuch as L-homoserine for growth (Japanese Patent Publication (Kokoku)Nos. 48-28078 and 56-6499), mutant strains with resistance to AEC andwhich further require an amino acid such as L-leucine, L-homoserine,L-proline, L-serine, L-arginine, L-alanine and L-valine (U.S. Pat. Nos.3,708,395 and 3,825,472), L-lysine producing mutant strains withresistance to DL-α-amino-ε-caprolactam, α-amino-lauryllactam, asparticacid analogue, sulfa drug, quinoid and N-lauroylleucine, L-lysineproducing mutant strains with resistance to oxaloacetate decarboxylaseor a respiratory tract enzyme inhibitor (Japanese Patent Laid-open Nos.50-53588, 50-31093, 52-102498, 53-9394, 53-86089, 55-9783, 55-9759,56-32995, 56-39778, Japanese Patent Publication Nos. 53-43591 and53-1833), L-lysine producing mutant strains which require inositol oracetic acid (Japanese Patent Laid-open Nos. 55-9784 and 56-8692),L-lysine producing mutant strains that are susceptible to fluoropyruvicacid or a temperature of 34° C. or higher (Japanese Patent Laid-openNos. 55-9783 and 53-86090), L-lysine producing mutant strains ofBrevibacterium or Corynebacterium bacteria with resistance to ethyleneglycol (U.S. Patent Application No. 333,455), and so forth.

Specific examples include, for example, the Brevibacteriumlactofermentum ATCC 31269, Brevibacterium flavum ATCC 21475, andCorynebacterium acetoglutamicum ATCC 21491 strains.

Furthermore, the Brevibacterium lactofermentum ATCC 13869/pVK-C*, plysEstrain described in the examples is also a preferred L-lysine producingcoryneform bacterium. This strain was obtained by incorporating aplasmid pVK-C* containing the gene coding for aspartokinase which isdesensitized to feedback inhibition by L-lysine and L-threonine (lysC*)and a plasmid plysE (U.S. Patent Application No. 2003113899) containingthe lysE gene which is homologous to the gene which promotes secretionof L-lysine known for the Corynebacterium bacteria (International PatentPublication 9723597A2) into the ATCC 13869 strain, which is a wild typestrain of Brevibacterium lactofermentum.

The lysC* gene can be isolated from, for example, the L-lysine producingmutant strain AJ3463 (FERM P-1987) (see Japanese Patent Publication No.51-34477) which is generated by mutagenesis of the ATCC 13869 strain.The AJ3463 strain was deposited at International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (formerly National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Address:Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,305-8566, Japan) on Mar. 22, 1973, and assigned accession number FERMP-1987. Furthermore, a lysC* gene fragment can also be isolated from theBrevibacterium lactofermentum AJ12691 strain which contains a plasmidp399AK9B containing the gene. The AJ12691 strain was deposited atInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology on Apr. 10, 1992, and assignedaccession number FERM P-12918. Then, it was converted to aninternational deposit under the provisions of the Budapest Treaty onFeb. 10, 1995, and assigned accession number FERM BP-4999. The plasmidp399AK9B (U.S. Pat. No. 5,766,925) was obtained by inserting a DNAfragment enabling autonomous replication of the plasmid inCorynebacterium bacteria into a plasmid p399AK9 which was obtained byinserting lysC derived from the AJ3463 strain into the cloning vectorpHSG399 (see Takeshita, S et al, Gene (1987), 61, 63-74).

In the aforementioned desensitized aspartokinase, the alanine residue atposition 279 of the α-subunit and the alanine residue at position 30 theβ-subunit of the wild-type aspartokinase are each replaced with athreonine residue. The α-subunit and the β-subunit are both encoded inthe same frame of the lysC gene. The nucleotide sequence of the lysC*gene and the amino acid sequence of the α-subunit of the desensitizedaspartokinase are shown in the sequence listing as SEQ ID NOS: 5 and 6,respectively, and the nucleotide sequence of the same gene and the aminoacid sequence of the β-subunit of the desensitized aspartokinase areshown as SEQ ID NOS: 7 and 8, respectively.

The lysE gene of coryneform bacteria can be obtained by PCR (polymerasechain reaction, see White, T. J. et al., Trends Genet., 5, 185 (1989))using primers based on the reported nucleotide sequence (GenBankaccession X96471), for example, primers shown as SEQ ID NOS: 3 and 4,and a chromosomal DNA of coryneform bacterium as the template. Anucleotide sequence of a DNA fragment containing the Corynebacteriumglutamicum lysG and lysE genes (GenBank accession X96471) is shown asSEQ ID NO: 10, and the amino acid sequence of the LysE protein encodedby this gene is shown as SEQ ID NO: 9. LysG is encoded by acomplementary sequence corresponding to the nucleotide numbers 1723 to2352 in SEQ ID NO: 8.

The DNAs coding for the α-subunit, β-subunit and LysE protein ofaspartokinase include DNAs coding for proteins that may includedeletions, substitutions, insertions or additions of one or severalamino acid residues at one or several positions in each protein,provided that the activities of the proteins are not lost. Although thenumber of amino acid residues meant by the term “several” may varydepending on the positions in the three dimensional structures of theproteins and types of amino acid residues, it is preferably 2 to 30,more preferably 2 to 20, particularly preferably 2 to 10, for eachprotein. This is based on the following reasons. That is, it is becausesome amino acids are highly homologous to each other, and thedifferences among such amino acids do not greatly affect the threedimensional structures and activities of proteins. Therefore, eachprotein may be one having a homology of 50% or more, preferably 70% ormore, more preferably 90% or more, particularly preferably 95% or more,to the amino acid residues of SEQ ID NO: 6, 8 or 10 and having theactivity of aspartokinase or LysE protein.

Such modification of the proteins as described above is a conservativemutation that maintains the activity of each protein. The substitutionis a change in which at least on residue in an amino acid sequence isremoved, and another residue is inserted there. Examples of substitutionof an amino acid residue for an original amino acid residue consideredas a conservative substitution include substitution of ser or thr forala, substitution of gln, his or lys for arg, substitution of glu, gln,lys, his or asp for asn, substitution of asn, glu or gln for asp,substitution of ser or ala for cys, substitution of asn, glu, lys, his,asp or arg for gln, substitution of asn, gln, lys or asp for glu,substitution of pro for gly, substitution of asn, lys, gln, arg or tyrfor his, substitution of leu, met, val or phe for ile, substitution ofile, met, val or phe for leu, substitution of asn, glu, gln, his or argfor lys, substitution of ile, leu, val or phe for met, substitution oftrp, tyr, met, ile or leu for phe, substitution of thr or ala for ser,substitution of ser or ala for thr, substitution of phe or tyr for trp,substitution of his, phe or trp for tyr, and substitution of met, ile orleu for val.

A DNA coding for substantially the same protein as the protein havingthe amino acid sequence as shown in SEQ ID NOS: 6, 8 or 10 can beobtained by modifying the nucleotide sequence coding for the amino acidsequence as shown in SEQ ID NOS: 6, 8 or 10 by using, for example,site-specific mutagenesis, so that substitution, deletion, insertion oraddition of one or several amino acid residues occurs. Such a modifiedDNA can be obtained in a conventional manner by treating with a regentor under conditions which cause a mutation. Examples of such a treatmentinclude treating the DNA coding for the protein of the present inventionwith hydroxylamine, ultraviolet ray irradiation of a microorganismcontaining the DNA, treating with a regent such asN-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid.

A DNA coding for such a modified protein as described above can also beobtained by isolating a DNA which is able to hybridize with the lysCgene, lysE gene or a portion of these genes under stringent conditionsand still encodes a protein having aspartokinase activity or theactivity of the LysE protein. The term “stringent conditions” includes acondition when a so-called specific hybrid is formed, and non-specifichybrid is not formed. The stringent conditions include, for example,conditions under which DNAs having high homology to each other, forexample, DNAs having a homology of not less than 70%, preferably notless than 80%, more preferably not less than 90%, particularlypreferably not less than 95%, are able to hybridize. The stringentconditions also include typical washing conditions of Southernhybridization, i.e., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, at60° C.

Examples of L-lysine producing bacteria belonging to the genusEscherichia include mutants having resistance to L-lysine analogues. TheL-lysine analogue inhibits growth of Escherichia bacteria, but thisinhibition is fully or partially eliminated when L-lysine coexists in amedium. Examples of L-lysine analogues include oxalysine, lysinehydroxamate, (S)-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine,α-chlorocaprolactam, and so forth. Mutants having resistance to theselysine analogues can be obtained by subjecting Escherichiamicroorganisms to a conventional artificial mutation treatment. Specificexamples of bacterial strains used for producing L-lysine include E.coli AJ11442 (FERM BP-1543, NRRL B-12185; see Japanese Patent Laid-openNo. 56-18596 and U.S. Pat. No. 4,346,170) and E. coli VL611 strains. TheAJ11442 strain was deposited at International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (formerly National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Address:Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,305-8566, Japan) on May 1, 1981, and assigned accession number FERMP-5084. Then, this deposit was converted to an international depositunder the provisions of the Budapest Treaty on Oct. 29, 1987, andassigned accession number FERM BP-1543. In these microorganisms,feedback inhibition of aspartokinase by L-lysine is desensitized.

Furthermore, for example, bacteria with enhanced expression of a genecoding for an enzyme involved in L-lysine biosynthesis other thandesensitized aspartokinase may also used as a preferred L-lysineproducing bacteria. Examples of such an enzyme include enzymes involvedin the diaminopimelate pathway, such as dihydrodipicolinate synthase,dihydrodipicolinate reductase, diaminopimelate decarboxylase,diaminopimelate dehydrogenase (International Patent PublicationWO96/40934 for all of the foregoing enzymes), phosphoenolpyruvatecarboxylase (Japanese Patent Laid-open No. 60-87788), aspartateaminotransferase (Japanese Patent Publication No. 6-102028),diaminopimelate epimerase (Japanese Patent Laid-open No. 2003-135066),and aspartate semialdehyde dehydrogenase (International PatentPublication WO00/61723), enzymes involved in the aminoadipate pathway,such as homoaconitate hydratase (Japanese Patent Laid-open No.2000-157276), and so forth.

Specific examples of E. coli strains having L-lysine producing abilityinclude the E. coli W3110(tyrA)/pCABD2 strain (International PatentPublication WO95/16042) and so forth. The E. coli W3110(tyrA)/pCABD2strain was obtained by introducing the plasmid pCABD2 containing genesencoding the L-lysine biosynthesis system enzymes into W3110(tyrA),which is a tyrA deficient strain of E. coli (it was designated asAJ12604, deposited at International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (formerlyNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Address: Tsukuba Central 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Jan. 28,1991, and assigned accession number FERM P-11975, and then the depositwas converted to an international deposit under the provisions of theBudapest Treaty on Sep. 26, 1991, and assigned accession number FERMBP-3579).

The plasmid pCABD2 contains a gene coding for a mutantdihydrodipicolinate synthase, wherein the histidine residue at position118 is mutated to a tyrosine residue, and feedback inhibition byL-lysine is desensitized, a gene coding for a mutant aspartokinase III,wherein threonine residue at position 352 is mutated to an isoleucineresidue, and feedback inhibition by L-lysine is desensitized, and genescoding for dihydrodipicolinate reductase and diaminopimelatedehydrogenase.

Furthermore, the E. coli W3110(tyrA) strain can be obtained as describedbelow. That is, many strains obtained by introducing a plasmid into theW3110(tyrA) strain are disclosed in European Patent Laid-openPublication No. 488424/1992. For example, a strain obtained byintroducing a plasmid pHATerm was designated as E. coliW3110(tyrA)/pHATerm strain, and deposited at the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, and assigned accession number FERM BP-3653. The W3110(tyrA)strain can be obtained by, for example, eliminating the plasmid pHATermfrom that E. coli W3110(tyrA)/pHATerm strain.

Elimination of the Plasmid can be Performed in a Conventional Manner.

Furthermore, the WC196 strain (see International Patent PublicationWO96/17930) can also be used as an L-lysine producing strain of E. coli.The WC196 strain was bred by imparting AEC (S-(2-aminoethyl)cysteine)resistance to the W3110 strain derived from E. coli K-12. This strainwas designated E. coli AJ13069, and deposited at the National Instituteof Bioscience and Human-Technology, Agency of Industrial Science andTechnology (presently International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Tsukuba Central6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan)) onDec. 6, 1994, and assigned accession number FERM P-14690. Then, thedeposit was converted to an international deposit under the provisionsof the Budapest Treaty on Sep. 29, 1995, and assigned accession numberFERM BP-5252.

The microorganism usable for the present invention may have decreasedactivity of an enzyme that catalyzes a reaction for generation ofcompounds other than L-lysine via pathway which branches off of thebiosynthetic pathway of L-lysine, or an enzyme which down regulatesL-lysine production, or may be deficient in such an enzyme. Illustrativeexamples of the enzyme involved in L-lysine production includehomoserine dehydrogenase, lysine decarboxylase (cadA, ldcC) and malicenzyme. Strains in which the activities of these enzymes are decreasedor deficient are described in International Patent PublicationsWO95/23864, WO96/17930, WO2005/010175, and so forth.

To reduce or eliminate enzymatic activities, genes encoding the enzymeson a chromosome may be mutated by a common mutagenesis method so thatintracellular activities of the enzymes are reduced or eliminated. Forexample, this can be achieved by using genetic recombination toeliminate these genes coding for the enzymes on a chromosome or tomodify an expression control sequence, such as a promoter or theShine-Dalgarno (SD) sequence. It can also be achieved by introducing anamino acid substitution (missense mutation), introducing a stop codon(nonsense mutation), introducing a frame shift mutation adding ordeleting one or two nucleotides into coding regions for the enzymes onthe chromosome, or deleting a part of the genes (Journal of BiologicalChemistry, 272:8611-8617 (1997)). The enzymatic activities can also bedecreased or eliminated by constructing a gene which encodes a mutantenzyme, wherein the coding region is deleted, and replacing the wildtype gene on the chromosome by homologous recombination or the like withthe mutated gene, or introducing a transposon or IS factor into thegene.

For example, the following methods may be employed to introduce amutation which causes a decrease in the activities of the aforementionedenzymes or eliminates the activities by genetic recombination. Theobjective gene on a chromosome can be replaced with a mutant gene whichcannot produce an enzyme that normally functions by modifying a partialsequence of the objective gene to prepare the mutant gene, andtransforming a coryneform bacterium with a DNA containing the mutantgene to cause recombination between the mutant gene and the gene on thechromosome. Such site-specific mutagenesis based on gene substitutionusing homologous recombination has been already established, and methodsusing linear DNA, methods using plasmids containing atemperature-sensitive replication origin (Proc. Natl. Acad. Sci. USA,2000, vol. 97, No. 12, pp. 6640-6645; U.S. Pat. No. 6,303,383; JapanesePatent Laid-open No. 05-007491) and so forth, are known. Furthermore,such site-specific mutagenesis based on gene substitution usinghomologous recombination as described above can also be performed with aplasmid which is not able to replicate in the host.

Furthermore, microorganisms which have been modified so that expressionamount of the L-lysine and L-arginine secretion gene, ybjE, is increasedcan also be used for the present invention (International PatentPublication WO2005/073390).

Examples of L-lysine producing bacteria belonging to the genus Serratiainclude Serratia bacteria transformed with a DNA coding fordihydrodipicolinate synthase which has a mutation that desensitizesfeedback inhibition by L-lysine, and Serratia bacteria containingaspartokinase which is desensitized to feedback inhibition by L-lysine(International Patent Publication WO96/41871).

Examples of coryneform bacteria which produce L-arginine includewild-type strains of coryneform bacteria: coryneform bacteria resistantto certain agents including sulfa drugs, 2-thiazolealanine,α-amino-β-hydroxyvaleric acid and so forth: coryneform bacteriaexhibiting auxotrophy for L-histidine, L-proline, L-threonine,L-isoleucine, L-methionine or L-tryptophan in addition being resistantto 2-thiazolealanine (Japanese Patent Laid-open No. 54-44096);coryneform bacteria resistant to ketomalonic acid, fluoromalonic acid,or monofluoroacetic acid (Japanese Patent Laid-open No. 57-18989);coryneform bacteria resistant to argininol (Japanese Patent Laid-openNo. 62-24075); coryneform bacteria resistant to X-guanidine (Xrepresents a derivative of fatty acid or aliphatic chain, JapanesePatent Laid-open No. 2-186995) and so forth. Furthermore, the coryneformbacteria which are deficient in the L-arginine repressor (U.S. PatentApplication No. 20020045233), and the coryneform bacteria with increasedglutamate dehydrogenase activity (European Patent Publication Laid-openNo. 1057893) are also suitable strains for L-arginine production.

Specifically, the examples include the Brevibacterium flavum AJ11169(FERM BP-6892), Corynebacterium glutamicum AJ12092 (FERM BP-6906),Brevibacterium flavum AJ11336 (FERM BP-6893), Brevibacterium flavumAJ11345 (FERM BP-6894), and Brevibacterium lactofermentum AJ12430 (FERMBP-2228) strains. The AJ11169 and AJ12092 strains are resistant to2-thiazolealanine (Japanese Patent Laid-open No. 54-44096). The AJ11336strain is resistant to argininol and sulfadiazine (Japanese PatentPublication No. 62-24075). The AJ11345 strain is resistant to arginino,2-thiazolealanine and sulfaguanidine, and is auxotrophic for histidine(Japanese Patent Publication No. 62-24075). The AJ12430 strain isresistant to octylguanidine and 2-thiazolealanine (Japanese PatentLaid-open No. 2-186995).

The Corynebacterium glutamicum AJ12092 was deposited at NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology (presently International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, 305-8566, Japan) on Dec. 6, 1994, and assigned accessionnumber FERM P-12092. Then, the deposit was converted to an internationaldeposit under the provisions of the Budapest Treaty on Oct. 1, 1999, andassigned accession number FERM BP-6906.

Examples of Escherichia bacteria which are able to produce L-arginineinclude E. coli transformed with the argA gene (see Japanese PatentLaid-open No. 57-5693), and E. coli strain 237 (Russian PatentApplication No. 2000117677), which is an L-arginine producing derivativeof mutant strain which is able to assimilate an acetic acid. The 237strain was deposited at the Russian National Collection of IndustrialMicroorganisms (VKPM), GN11 Genetika (Address: Russia, 117545, Moscow, 1Dorozhnyproezd, 1) on Apr. 10, 2000, and assigned number VKPM B-7925.The deposit was converted to an international deposit under theprovisions of the Budapest Treaty on May 18, 2001. The E. coli strain382 is a mutant which is resistant to feedback inhibition by L-arginine(Japanese Patent Laid-open No. 2002-017342), which is a derivative ofthe 237 strain, and can also be employed. The E. coli 382 strain wasdeposited at Russian National Collection of Industrial Microorganisms(VKPM) with a number VKPM B-7926 on Apr. 10, 2000, and the deposit wasconverted to an international deposit under the provisions of theBudapest Treaty on May 18, 2001.

Examples of Serratia bacteria which are able to produce L-arginineinclude Serratia marcescens which is unable to decompose L-arginine andis resistant to an arginine antagonist and canavanine, and isauxotorophic for lysine (see Japanese Patent Laid-open No. 52-8729).

Examples of coryneform bacteria which are able to produce L-histidineinclude microorganisms belonging to the genus Brevibacterium which areresistant to a thiamin antagonist, specifically, Brevibacteriumlactofermentum FERM P-2170, FERM P-2316, FERM P-6478, FERM P-6479, FERMP-6480 and FERM P-6481 strains (Japanese Patent Laid-open PublicationNo. 59-63194). Furthermore, the examples include mutant strainsbelonging to the genus Brevibacterium or Corynebacterium which areresistant to polyketides and L-histidine producing ability,specifically, the FERM P-4161, FERM P-7273, FERM P-8371, FERM P-8372 andATCC 14067 strains.

Examples of Escherichia bacteria which are able to produce L-histidineinclude mutant strains belonging to the genus Escherichia which areresistant to a histidine analogue, for example, the E. coli R-344strain, and Escherichia bacteria transformed with L-histidine synthesissystem enzyme genes isolated from the strain R-344. Specifically, theexamples include the E. coli NRRL-12116, NRRL-12118, NRRL-12119,NRRL-12120 and NRRL-12121 strains (Japanese Patent Laid-open No.56-5099).

Examples of Bacillus bacteria able to produce L-histidine include mutantstrains belonging to the genus Bacillus which are resistant to ahistidine analogue, and Bacillus bacteria transformed with a geneobtained from these mutant strains which are involved in resistance tohistidine antagonist. Specifically, the examples include the FERMBP-218, FERM BP-224 and FERM BP-219 strains (Japanese Patent Laid-openNo. 58-107192).

The fermentation broth or the processed product thereof containing thebasic substance obtained by the present invention will contain carbonateions or bicarbonate ions as counter anions for the dissociated basicsubstance. These carbonate ions or bicarbonate ions are emitted ascarbon dioxide gas when the culture medium is heated or concentrated, orif the pH of the medium is lowered by adding a strong acid such ashydrochloric acid. The relative amount of the basic substance among thesolid components in the fermentation broth is thus increased.

According to the present invention, by using bicarbonate ions, carbonateions or the like in place of chloride ions and sulfate ions, the amountof the chloride ions can be reduced even to a level not causingcorrosion of equipments, or sulfate ions can be reduced. Furthermore,after the fermentation, bicarbonate ions and carbonate ions can bereplaced with chloride ions only by adding hydrochloric acid to themedium, lysine hydrochloride can be obtained only by furtherconcentrating the medium without using ion exchange, and further,crystals of lysine hydrochloride can be directly separated.

In the present invention, the “fermentation product” includesconcentrate and dried product obtained from the fermentation broth, andproducts obtained by processing the fermentation broth or dried productthereof.

EXAMPLES

Hereafter, the present invention will be explained more specificallywith reference to the following examples.

Example 1 Construction of L-Lysine Producing Coryneform Bacterium

A gene coding for desensitized aspartokinase and a gene coding for alysine secretion factor were introduced into a wild coryneform bacteriumto prepare an L-lysine producing bacterium.

(1) Acquisition of Gene Coding for Desensitized Aspartokinase

A gene (lysC*) coding for aspartokinase (Ask*) which is desensitized tofeedback inhibition by L-lysine and L-threonine was isolated by PCR froman L-lysine producing mutant strain, AJ3463 (FERM P-1987, see JapanesePatent Publication No. 51-34477) derived from the Corynebacteriumglutamicum (Brevibacterium lactofermentum) ATCC 13869 strain bymutagenesis.

The AJ3463 strain was cultured in CM-Dex medium, and chromosomal DNA wasextracted from the obtained cells by a typical method (Biochem. Biophys.Acta., 72, 619-629 (1963)). By using this chromosomal DNA as a templatewith an oligonucleotide ASK-F (SEQ ID NO: 1) for introducing arestriction enzyme BamHI site at the 5′ end of the objective DNAfragment and an oligonucleotide ASK-R (SEQ ID NO: 2) for introducing arestriction enzyme KpnI site at the 3′ end of the objective DNA fragmentas primers for PCR, a gene DNA fragment containing lysC* as theobjective gene was amplified. For amplification, a cycle consisting of adenaturation step at 98° C. for 10 seconds, an annealing step at 55° C.for 30 second and an extension step at 72° C. for 2 minutes was repeated25 times. Enzyme, Pyrobest DNA Polymerase (Takara Shuzo), was usedaccording to the manufacturer's instructions.

The amplified DNA fragment was purified by a phenol/chloroform treatmentand ethanol precipitation, and then digested with the restrictionenzymes BamHI and KpnI. The obtained reaction mixture was developed byagarose gel electrophoresis, the band containing the lysC* gene wasexcised, and the gene fragment was purified by conventional methods.

A shuttle vector for E. coli and Corynebacterium glutamicum, pVK7 (seeU.S. Pat. No. 6,004,773), was separately treated with the restrictionenzymes BamHI and KpnI in a similar manner, and ligated to theaforementioned lysC* fragment. Competent cells of the E. coli JM109strain (Takara Shuzo) were transformed with the ligation reactionmixture according to the manufacturer's protocol, and severalkanamycin-resistant colonies were selected.

The pVK7 was constructed by ligating a cryptic plasmid of Brevibacteriumlactofermentum, pAM330, to a vector for E. coli, pHSG299 (Km^(r), seeTakeshita, S. et al., Gene, 61, 63-74, (1987)) as follows (see JapanesePatent Laid-open No. 11-266881, International Patent PublicationWO99/07853). pAM330 was prepared from the Brevibacterium lactofermentumATCC 13869 strain. pHSG299 was digested with AvaII (Takara Shuzo), whichhad been blunt-ended with T4 DNA polymerase, then digested with HindIII(Takara Shuzo), and ligated to pAM330 blunt-ended with T4 DNApolymerase. Thus, pVK7 was obtained. pVK7 is autonomously replicable incells of E. coli and Brevibacterium lactofermentum, and contains amultiple cloning site derived from pHSG299, lacZ′, and a kanamycinresistance gene as a marker.

Plasmid DNAs were extracted from the kanamycin-resistant coloniesobtained as described above in a conventional manner, and the plasmidcontaining the objective lysC* gene was designated pVK-C*.

(2) Acquisition of a Gene Coding for Lysine Secretion Factor lysE

By using chromosomal DNA from Brevibacterium lactofermentum ATCC 13869as a template, the lysE gene was isolated by PCR (see U.S. PatentApplication No. 2003113899). The lysE gene has been known to function inCorynebacterium bacteria to promote secretion of L-lysine (InternationalPatent Publication 9723597A2). The chromosomal DNA of the strain wasprepared in the same manner as described above.

LysE-F (SEQ ID NO: 3) and LysE-R (SEQ ID NO: 4) were used as theprimers. The PCR was performed using Pyrobest (Takara Shuzo) with a heattreatment at 94° C. for 90 seconds, and the following cycle was repeated30 times: denaturation at 94° C. for 20 seconds, annealing at 55° C. for30 seconds and extension reaction at 72° C. for 60 seconds. Then thereaction was incubated at 72° C. for 10 minutes. A DNA fragment of thepredicted size was obtained by this reaction. This DNA fragment waspurified, and then cloned into cloning vector pCR2.1 (Invitrogene)according to the manufacturer's protocol. Competent cells of the E. coliJM109 strain (Takara Shuzo) were transformed with the ligation reactionmixture according to the manufacturer's protocol, and severalampicillin-resistant colonies were selected. Plasmid DNAs were extractedfrom these colonies, and the plasmid having the desired structure wasdesignated pCRlysE.

Then, pCRlysE was digested with the restriction enzymes BamHI and XbaI,and subjected to agarose gel electrophoresis to obtain a fragmentcontaining the lysE gene. A shuttle vector for E. coli andCorynebacterium glutamicum, pKC (see U.S. Patent application No.2003113899), was separately treated with the restriction enzymes BamHIand KpnI in a similar manner, and subjected to agarose gelelectrophoresis to obtain a gene fragment containing the chloramphenicolresistance gene. This gene was purified and then ligated to theaforementioned lysE fragment. By using this ligation reaction mixture,competent cells of the E. coli JM109 strain (Takara Shuzo) weretransformed according to the manufacturer's protocol, and severalchloramphenicol-resistant colonies were chosen. Plasmids were preparedfrom the colonies obtained as described above to obtain the LysEexpression plasmid, plysE.

pKC4 was prepared as follows. A plasmid pHK4 (see Japanese PatentLaid-open No. 5-7491) having a replication origin derived from thealready obtained plasmid pHM1519, which is autonomously replicable incoryneform bacteria (Agric. Biol. Chem., 48, 2901-2903 (1984)), wasdigested with the restriction enzymes BamHI and KpnI to obtain a genefragment containing the replication origin. The obtained fragment wasblunt-ended with a DNA Blunting Kit (Takara Shuzo), and inserted at theKpnI site of pHSG399 (Takara Shuzo) by ligation using a KpnI Linker(Takara Shuzo). Competent cells of the E. coli JM109 strain (TakaraShuzo) were transformed with this ligation reaction mixture according tothe manufacturer's protocol, and several chloramphenicol-resistantcolonies were selected. Plasmids were prepared from the coloniesobtained as described above to obtain pKC4.

(3) Construction of L-Lysine Producing Coryneform Bacterium

The above-described two plasmids, pVK-C* and plysE, were introduced intothe Brevibacterium lactofermentum ATCC 13869 strain by electroporation.The electroporation was performed by using Gene Pulser (BIO-RAD). Thedistance between the electrodes in the cuvette was 0.1 cm and theelectric pulse application conditions were 25 μF, 200Ω and 1.8 kV. Thestrains containing the plasmids were selected on a CM-Dex agar plate(see below for the composition of the medium) containing 5 μg/l ofchloramphenicol and 25 μg/l of kanamycin. The strain containing theplasmid was cultured overnight at 31.5° C. with shaking in the CM-Dexliquid medium containing 5 μg/l of chloramphenicol and 25 μg/l ofkanamycin. The culture was performed in 3 ml of the culture medium in atest tube with shaking.

The CM-Dex medium was prepared as follows. All the components listed inTable 1 were mixed, adjusted to pH 7.5 with KOH, and then sterilized byautoclaving at 120° C. for 20 minutes. In the agar medium, agar wasadded to a final concentration of 20 g/L.

TABLE 1 Composition of CM-Dex medium (per 1 L) Glucose 5 g Polypeptone10 g Yeast extract 10 g KH₂PO₄ 1 g MgSO₄•7H₂O 0.4 g FeSO₄•7H₂O 0.01 gMnSO₄•4H₂O or 5H₂O 0.01 g Urea 3 g Mameno (soy bean protein hydrolysate,1.2 g in terms of nitrogen weight) Biotin 10 μg (Filled to 1 L withsterilized water)

As described above, an L-lysine producing coryneform bacterium, ATCC13869/pVK-C*, plysE was obtained.

Example 2 Growth of L-Lysine Producing Bacterium in an Alkaline Medium,and Effect of the Total Ammonia Concentration on L-Lysine Production

By using the L-lysine producing bacterium constructed in Example 1, thetotal ammonia concentration not inhibiting productivity of L-lysine inan alkaline medium was investigated.

First, a conventional culture method was used. That is, a medium (mediumB) obtained by adding ammonium sulfate was added to the medium A(composition was shown in Table 2) in an amount of 55% (w/w) based onglucose was used. The pH of the medium was maintained constant withammonia gas during the culture, to perform L-lysine fermentation. The pHwas controlled to be 7.0 or 8.0.

TABLE 2 Composition medium A (per 1 L) Glucose 100 g Yeast extract 10 gKH₂PO₄ 1 g MgSO₄•7H₂O 1 g Vitamin B1 hydrochloride 2 mg Biotin 0.5 mgNicotinamide 5 mg FeSO₄•7H₂O 10 mg MnSO₄•4H₂O or 5H₂O 10 mg 10% GD-113(antifoaming agent) 0.05 mL

Specifically, the culture was performed as follows. The aforementionedstrain was inoculated into 3 ml of the CM-Dex liquid medium and culturedovernight at 31.5° C. with shaking, and 200 μl of the medium wasuniformly spread onto CM-Dex agar medium. The culture was performedovernight at 31.5° C. as a stationary culture. Then, one third of theL-lysine producing bacterial cells which grew on the agar medium on oneplate were inoculated to 300 ml of medium B in a jar fermenter andcultured. During the culture, the medium was aerated with 300 ml perminute of filter-sterilized air, the stifling rate was maintained at 700rpm, and the temperature of the medium was maintained at 31.5° C. Theresults are shown in FIG. 1.

As a result, at pH 7.0, 17.4 g/L of L-lysine was accumulated, and theproduction rate was 0.725 g/L/hr. On the other hand, at pH 8.0, thegrowth of cells was almost not existent, and fermentation did notprogress (FIG. 1).

Then, fermentation was performed using the L-lysine producing bacteriain a medium without ammonium sulfate.

The aforementioned strain was inoculated into 3 ml of the CM-Dex liquidmedium and cultured overnight at 31.5° C. with shaking, and 200 μl ofthe medium was uniformly spread onto the CM-Dex agar medium and leftovernight at 31.5° C. 300 ml of medium A (without ammonium sulfate) wasplaced in a jar fermenter, and the pH was adjusted to 7.8, 8.2, or 8.9by bubbling ammonia gas through the medium. One third of the L-lysineproducing bacterial cells which grew on the agar medium on one platewere inoculated into the medium, and cultured. During the culture, themedium was aerated with 300 ml per minute of filter-sterilized air, thestifling rate was maintained at 700 rpm, and the temperature of themedium was maintained constant at 31.5° C. During the culture, aconstant pH was maintained by bubbling ammonia gas through the medium.As a result, it was confirmed that, at pH 8.9, after the total ammoniaconcentration in the medium exceeded 100 mM, growth and L-lysineproduction, in particular, were strongly inhibited.

In the aforementioned culture method, carbon dioxide gas generated bythe L-lysine producing bacteria dissolved in the medium as carbonateions or bicarbonate ions, which results in a lowered pH as the cultureprogresses. Therefore, the amount of added ammonia necessary to controlthe pH at the predetermined level increases. Furthermore, the pH levelto which the medium had been adjusted became higher, the concentrationsof dissolved carbonate ions and bicarbonate ions became higher, andtherefore the concentration of the ammonia added in order to adjust thepH to the predetermined value became higher.

Non-dissociated ammonia easily penetrates cells resulting in cellulardamage the cells. Since a higher pH results in a lower amount ofdissociation of ammonia, the growth of bacteria was inhibited as the pHincreases, even if the total ammonia concentration is maintained at aconstant level. Therefore, it was concluded that at pH 8.9 or lower, ifthe total ammonia concentration is controlled to be low, for example, at100 mM or lower, inhibition of bacterial growth and L-lysineaccumulation is not significant.

Then, culture for L-lysine production was performed while the totalammonia concentration in the medium was controlled at 100 mM or lower atpH 7.8, 8.2, and 8.9.

The aforementioned strain was inoculated into 3 ml of the CM-Dex liquidmedium and cultured overnight at 31.5° C. with shaking, and 200 μl ofthe medium was uniformly spread onto the CM-Dex agar medium and leftovernight at 31.5° C. 300 ml of medium A (without ammonium sulfate) wasplaced in a jar fermenter, and the pH was adjusted to 7.5, 7.8, 8.2, or8.9 by bubbling ammonia gas through the medium. One third of theL-lysine producing bacterial cells which grew on the agar medium on oneplate were inoculated into the medium, and cultured. During the culture,the medium was aerated with 300 ml per minute of filter-sterilized air,the stirring rate was maintained at 700 rpm, and the temperature of themedium was maintained at 31.5° C. During the culture, the pH wasmaintained at each of the predetermined levels with 6 N potassiumhydroxide instead of ammonia.

The total ammonia concentration in the medium was measured by using anammonia electrode and an ion meter (Orion). The medium was periodicallysampled, and the total ammonia concentration controlled to within 0 to100 mM by adding 10% aqueous ammonia solution as required. Furthermore,by monitoring the dissolved oxygen concentration in the medium, a sharpincrease in the dissolved oxygen concentration when ammonia was depletedwas detected. When this occurred, a 10% aqueous ammonia solution wasadded to prevent continuous depletion of ammonia in the medium. Theresults are shown in FIG. 2.

As a result, favorable growth and L-lysine production were observed atall pH levels from 7.8 to 8.9. Compared with the fermentation performedat pH 7.0 using the conventional culture method, the production rate of115% or higher was observed.

Example 3 Production of L-Lysine

In this example, L-lysine fermentation was performed by controlling onlythe total ammonia concentration, but not controlling the pH. The rangeof the total ammonia concentration was maintained at 100 mM or lower.This range was chosen based on the results in Example 2.

The aforementioned strain was inoculated into 3 ml of the CM-Dex liquidmedium and cultured overnight at 31.5° C. with shaking, and 200 μl ofthe medium was uniformly spread onto the CM-Dex agar medium and leftovernight at 31.5° C. 300 ml of medium A (without ammonium sulfate) wasplaced in a jar fermenter, and the total ammonia concentration of themedium was adjusted to 23.8 mM by bubbling ammonia gas through themedium. One third of the L-lysine producing bacterial cells which grewon the agar medium on one plate were inoculated into the medium, andcultured. During the culture, the medium was aerated with 300 ml perminute of filter-sterilized air, the stirring rate was maintained at 700rpm, and the temperature of the medium was maintained at 31.5° C. Thetotal ammonia concentration was measured periodically, and anappropriate amount of 10% aqueous ammonia was added to the medium asrequired so that the total ammonia concentration was maintained between0 to 100 mM. As a result, 15.9 g/L of lysine was accumulated, andL-lysine fermentation progressed (FIG. 3).

Example 4 Construction of L-Lysine Producing E. coli Bacterium

<1> Construction of Strain in which the cadA and ldcC Genes Coding forLysine Decarboxylase are Disrupted

A lysine decarboxylase deficient strain was constructed first. Lysinedecarboxylases are encoded by the cadA gene (Genbank Accession No.NP_(—)418555, SEQ ID NO: 15), and the ldcC gene (Genbank Accession No.NP_(—)414728, SEQ ID NO: 17) (see International Patent PublicationWO96/17930). In this example, the WC196 strain was used as the parentstrain.

The cadA and ldcC genes coding for lysine decarboxylase deleted by usingthe method developed first by Datsenko and Wanner called “Red-drivenintegration” (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, pp.6640-6645), and an excision system derived from λ phage (J. Bacteriol.,2002 September, 184 (18):5200-3, Interactions between integrase andexcisionase in the phage lambda excisive nucleoprotein complex, Cho E H,Gumport R I, Gardner J F). According to the “Red-driven integration”method, a PCR product obtained using synthetic oligonucleotide primersin which a part of the objective gene is designed on the 5′ side and apart of an antibiotic resistance gene is designed on the 3′ side, can beused to obtain a gene-disrupted strain in one step. Furthermore, byusing the excision system derived from λ phage in combination, theantibiotic resistance gene which had been incorporated into thegene-disruption strain can be eliminated (Japanese Patent Laid-open No.2005-058227).

(1) Disruption of the cadA Gene

The plasmid pMW118-attL-Cm-attR (Japanese Patent Laid-open No.2005-058827) was used as a template in PCR. pMW118-attL-Cm-attR wasobtained by inserting the attL and attR genes which are the attachmentsites of λ phage, and the cat gene which is an antibiotic resistancegene into pMW118 (Takara Bio). The order of insertion is attL-cat-attR.

PCR was performed with the synthetic oligonucleotide primers shown asSEQ ID NOS: 11 and 12 which have sequences corresponding to both ends ofattL and attR at the 3′ ends of the primers, and a sequencecorresponding to a portion of the objective cadA gene at the 5′ ends ofthe primers.

The amplified PCR product was purified on agarose gel and introduced byelectroporation into the E. coli WC169 strain which contains plasmidpKD46 which is temperature sensitive replicable. The plasmid pKD46(Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, pp. 6640-6645)contains a DNA fragment of 2154 nucleotides in total of λ phagecontaining genes coding for Red recombinase of the λ Red homologousrecombination system (λ, β, exo genes) controlled by arabinose-inducibleParaB promoter (GenBank/EMBL Accession No. J02459, nucleotides atpositions 31088 to 33241). The plasmid pKD46 is required to incorporatethe PCR product into the chromosome of the WC196 strain.

Competent cells for the electroporation were prepared as follows. Thatis, the E. coli WC196 strain cultured overnight at 30° C. in the LBmedium containing 100 mg/L of ampicillin was diluted 100 times with 5 mLof the SOB medium (Molecular Cloning: A Laboratory Manual, 2nd Edition,Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989))containing ampicillin (20 mg/L) and L-arabinose (1 mM). The cells in thediluted culture were grown at 30° C. with aeration until the OD600reached about 0.6, and then the culture was concentrated 100 times andwashed three times with 10% glycerol so that the cells could be used forelectroporation. The electroporation was performed by using 70 μl of thecompetent cells and about 100 ng of the PCR product. After theelectroporation, the cells were added to 1 mL of the SOC medium(Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook, J. etal., Cold Spring Harbor Laboratory Press (1989)), cultured at 37° C. for2.5 hours, and then cultured at 37° C. as plate culture on L agar mediumcontaining 25 mg/L of Cm (chloramphenicol), and Cm-resistantrecombinants were selected. Then, in order to remove the pKD46 plasmid,the cells were subcultured twice at 42° C. on the L agar mediumcontaining Cm, and ampicillin resistance of the colonies was examined toobtain an ampicillin-sensitive strain without pKD46.

Deletion of the cadA gene in the mutant identified on the basis of thechloramphenicol resistance gene was confirmed by PCR. The obtained cadAdeficient strain was designated WC196ΔcadA::att-cat strain.

Then, in order to remove the att-cat gene which had been introduced intothe cadA gene, a helper plasmid pMW-intxis-ts (Japanese Patent Laid-openNo. 2005-058827) was used. pMW-intxis-ts carries a gene coding forintegrase (Int) and a gene coding for excisionase (Xis) of λ phage, andis temperature sensitive replicable.

Competent cells of the WC196ΔcadA::att-cat strain obtained above wereprepared in a conventional manner, transformed with the helper plasmidpMW-intxis-ts, and cultured on L-agar medium containing 50 mg/L ofampicillin at 30° C. as a plate culture, and ampicillin-resistantstrains were selected.

Then, in order to remove the pMW-intxis-ts plasmid, the selected strainswere subcultured twice on L-agar medium at 42° C., and ampicillinresistance and chloramphenicol resistance of the obtained colonies wereexamined to obtain a chloramphenicol- and ampicillin-sensitivecadA-disrupted strain without att-cat and pMW-intxis-ts. This strain wasdesignated WC196ΔcadA.

(2) Deletion of the ldcC Gene from the WC196ΔcadA Strain

The ldcC gene was deleted from the WC196ΔcadA strain according to theaforementioned method using the primers of SEQ ID NOS: 13 and 14 asprimers for disruption of ldcC. A cadA- and ldcC-disrupted strain,WC196ΔcadAΔldcC, was thereby obtained.

<2> Introduction of Plasmid for Lys Production into WC196ΔcadAΔldcCStrain

The WC196ΔcadAΔldcC strain was transformed with plasmid pCABD2 for Lysproduction carrying the dapA, dapB and lysC genes (International PatentPublication WO01/53459) in a conventional manner to obtainWC196ΔcadAΔldcC/pCABD2 strain (WC196LC/pCABD2).

Example 5 L-Lysine Production Using E. coli

This example shows an example of the present invention applied to theproduction of L-lysine by E. coli. In this example, L-lysine wasproduced by fermentation without adding ammonium sulfate and ammoniumchloride, which are generally added to the media for the purpose ofsupplying nitrogen and counter ions for L-lysine in L-lysine productionby fermentation. Specifically, the culture was performed withoutcontrolling the pH, but controlling the ammonia concentration in themedium. The range within which the ammonia concentration in the mediumshould be controlled was examined beforehand. As a result, it wasconfirmed that the total ammonia concentration is preferably in therange of 50 to 100 mM. Therefore, in the practical main culture, thetotal ammonia concentration was controlled to be 100 mM or lower bybubbling ammonia gas. Furthermore, when the total ammonia concentrationdecreased to 50 mM, it was controlled with ammonia gas to maintain thatconcentration.

WC196ΔcadAΔldcC/pCABD2 was used for lysine production. 300 ml of theL-lysine production medium for E. coli shown in Table 3 placed in a jarfermenter was used. The total ammonia concentration was adjusted to 95mM by bubbling ammonia gas To this medium, cells obtained by culturingthe L-lysine producing strain on the entire surface of LB agar mediumcontaining 20 μg/L of streptomycin and culturing it at 37° C. for 24hours were inoculated. The amount of the inoculated cells correspondedto the cells grown on three plates of the agar medium. The culture wasperformed with a temperature of the medium maintained at 37° C.,aeration of 50 ml per minute of filter-sterilized air, and a stirringrate of 700 rpm. When the dissolved oxygen concentration in the mediumdecreased to 20% saturation, the aeration rate was changed to 100 mL perminute. The feed solution for E. coli shown in Table 4 was appropriatelyadded dropwise to the medium so that glucose is not depleted, and theconcentration thereof does not become 30 g/L or higher in the medium.Finally, when 36 g of glucose was consumed, the culture was terminated.As a result, the culture could be favorably performed so that all theadded glucose was consumed after 33 hours, 13.4 g of L-lysine wasaccumulated, and the production rate of L-lysine was 1.2 g/L/hr. Theyield of this production was 37%.

As a control, results are shown for L-lysine production performed withthe same strain by adding ammonium sulfate, and not controlling thetotal ammonia concentration, but controlling the pH, similar to commonproduction methods for basic amino acids. The same strain was inoculatedin a similar manner to a medium consisting of the L-lysine productionmedium for E. coli shown in Table 3 added with 13 g/L of ammoniumsulfate, and cultured while controlling the pH to be constant at 6.7 byappropriately bubbling ammonia gas. The culture temperature, aerationrate, and stirring rate were the same as those described above. In thiscase, the feed solution for E. coli added with 112.5 g/L of ammoniumsulfate (containing ammonium sulfate, Table 5) was added instead of thefeed solution for E. coli shown in Table 4, so that that glucose is notdepleted, and glucose concentration should not become 30 g/L or higherin the medium, and finally 36 g of glucose was consumed. As a result,all the added glucose was consumed after 33 hours, 14.7 g of L-lysinewas accumulated, and the production rate of L-lysine was 1.3 g/L/hr. Theyield of this production was 40%.

All the lysine concentrations described above are shown in terms oflysine hydrochloride. Furthermore, changes in the total ammoniaconcentration and pH during the cultures are shown in FIG. 4. Fromcomparison of these results, it was confirmed that, when the method ofthe present invention was used, L-lysine production by fermentationcould be performed without adding ammonium sulfate or ammonium chlorideat a production rate of about 92%, a yield of about 93% and an L-lysineproduction amount of about 91% compared with those obtained in thecommon fermentative production in which ammonium sulfate was added.

TABLE 3 Composition of L-lysine production medium for E. coli (per 1 L)glucose 30 g KH₂PO₄ 1 g MgSO₄•7H₂O 1.2 g Mameno (soy bean proteinhydrolysate, 0.77 g in terms of nitrogen weight) FeSO₄•7H₂O 30 mgMnSO₄•4H₂O or 5H₂O 30 mg p-aminobenzoic acid 2 mg L-threonine 300 mgDL-methionine 200 mg cystine 150 mg betaine 3.5 g GD-113 (antifoamingagent) 0.05 mL

Glucose and FeSO₄.7H₂O were weighed as a portion A, the other componentswere weighed as a portion B, and the portion A as it was and the portionB adjusted to pH 5.0 were separately sterilized by autoclaving at 115°C. for 10 minutes, and then mixed. 20 μg/L of streptomycin was added tothe medium prior to use.

TABLE 4 Composition of feed solution for E. coli (per 1 L) glucose  561g GD-113   7 μl KH₂PO₄ 1.48 g L-thr 0.44 g

The components were sterilized by autoclaving at 120° C. for 20 minutes.

20 μg/L of streptomycin was added to the medium prior to use.

TABLE 5 Composition of feed solution for E. coli containing ammoniumsulfate (per 1 L) glucose 561 g GD-113 7 μl KH₂PO₄ 1.48 g L-thr 0.44 g(NH₄)₂SO₄ 112.5 g/L

The components were sterilized by autoclaving at 120° C. for 20 minutes.

20 μg/L of streptomycin was added to the medium prior to use.

Example 6 Production of L-Arginine

This example shows an example of the present invention applied toL-arginine production by a coryneform bacterium. Corynebacteriumglutamicum AJ12092 (FERM BP-6906) was used as the L-arginine-producingstrain.

First, as a control, results are shown for L-arginine productionperformed with the same strain by adding ammonium sulfate, and notcontrolling the total ammonia concentration, but controlling the pH,similar to a common methods for production of a basic amino acid. Amedium for L-arginine production having the composition shown in Table6, with the addition of 65 g/L of ammonium sulfate, Also, the glucoseconcentration was changed to 40 g/L. 300 ml of this medium was placed ina jar fermenter, and the pH was controlled to be 7.0 by bubbling ammoniagas. To this medium, two plates of cells obtained by culturing theCorynebacterium glutamicum AJ12092 strain on the entire surface ofCM-Dex agar medium at 31.5° C. for 24 hours were inoculated. The culturewas performed at a temperature of the medium maintained at 31.5° C. withaeration of 150 ml per minute of filter-sterilized air and stifling at arate of 700 rpm. Furthermore, during the culture, the pH was controlledto be 6.9 by adding a 6 N KOH solution which had been separatelysterilized. As the culture progresses, the glucose concentrationdecreases. In order to maintain the glucose concentration at 30 to 40g/L, a separately sterilized glucose solution of 692 g/L wasappropriately added. The culture was performed for 54 hours. As aresult, 23.4 g/L of L-arginine was accumulated, the production yield ofL-arginine was 26.7% of the consumed glucose, and the production ratewas 0.43 g/L/hr. The amount of glucose consumed during the culture was29.1 g per batch.

Production of L-arginine when ammonium sulfate is not added to themedium, and while controlling only the total ammonia concentration, butnot the pH was performed. The results are shown hereinafter. 300 ml ofthe L-arginine production medium having the composition shown in Table 6but not containing ammonium sulfate was placed in a jar fermenter, andthe total ammonia concentration was adjusted to 12.6 mM by bubblingammonia gas. To this medium, two plates of cells of the L-arginineproducing strain cultured in the same manner as that of the control wereinoculated. The culture was performed in the same manner as that of thecontrol with maintaining the temperature at 31.5° C., aerating 150 mlper minute of filter-sterilized air, and maintaining the stirring rateat 700 rpm. By measuring the total ammonia concentration of the mediumperiodically or using an ammonia concentration controlling apparatus,the total ammonia in the medium was controlled so that it was at variouslevels during the culture. As a result, it was confirmed that the totalammonia concentration in the medium controlled to be about 20 mM byadding ammonia gas as required provided favorable results. The cultureperformed with controlling the total ammonia concentration in the mediumto be about 20 mM based on the above result favorably progressed, 24.2g/L of L-arginine was accumulated after 51 hours, and thus L-argininefermentation was attained (FIG. 4). The glucose consumed during theculture was 35.1 g per batch, the production yield of L-arginine was20.6% of the consumed glucose, and the production rate was 0.47 g/L/hr.Furthermore, the pH of the medium increased from 7.92 at the start ofthe culture to 8.02 at the end of the culture.

From the comparison of these results with those of the controlexperiment, it was demonstrated that the L-arginine production byfermentation could be performed without adding ammonium sulfate orammonium chloride at a yield of about 77% and a production rate higherby about 9% compared with those obtained in the common fermentativeproduction in which ammonium sulfate was added.

TABLE 6 Composition of L-arginine production medium (per 1 L) glucose150 g KH₂PO₄ 1 g MgSO₄•7H₂O 0.4 g Mameno (soy bean protein hydrolysate,0.23 g in terms of nitrogen weight) vitamin B1 hydrochloride 0.5 mgbiotin 0.5 mg FeSO₄•7H₂O 10 mg MnSO₄•4H₂O or 5H₂O 10 mg GD-113(antifoaming agent) 0.05 mL

The medium was adjusted to pH 7.0 with potassium hydroxide aqueoussolution, up to 1 L, and sterilized by autoclaving at 115° C. for 10minutes.

Explanation of Sequence Listing

SEQ ID NO: 1: Primer sequence for cloning of lysC gene

SEQ ID NO: 2: Primer sequence for cloning of lysC gene

SEQ ID NO: 3: Primer sequence for cloning of lysE gene

SEQ ID NO: 4: Primer sequence for cloning of lysE gene

SEQ ID NO: 5: Nucleotide sequence of lycC* gene and amino acid sequenceof α-subunit of inhibition-desensitized aspartokinase

SEQ ID NO: 6: Amino acid sequence of α-subunit ofinhibition-desensitized aspartokinase

SEQ ID NO: 7: Nucleotide sequence of lycC* gene and amino acid sequenceof β-subunit of inhibition-desensitized aspartokinase

SEQ ID NO: 8: Amino acid sequence of β-subunit ofinhibition-desensitized aspartokinase

SEQ ID NO: 9: Nucleotide sequence of lysE gene and amino acid sequenceof LysE protein

SEQ ID NO: 10: Amino acid sequence of LysE protein

SEQ ID NO: 11: Primer for disruption of cadA gene

SEQ ID NO: 12: Primer for disruption of cadA gene

SEQ ID NO: 13: Primer for disruption of ldc gene

SEQ ID NO: 14: Primer for disruption of ldc gene

SEQ ID NO: 15: Nucleotide sequence of cadA gene and amino acid sequenceof lysine decarboxylase

SEQ ID NO: 16: Amino acid sequence of lysine decarboxylase (cadA)

SEQ ID NO: 17: Nucleotide sequence of ldc gene and amino acid sequenceof lysine decarboxylase

SEQ ID NO: 16: Amino acid sequence of lysine decarboxylase (ldc)

INDUSTRIAL APPLICABILITY

According to the present invention, a basic substance can be produced byfermentation even at a high pH, which enables reduction of the amountsof industrial raw materials such as ammonium sulfate, withoutsubstantially degrading the performances essentially obtained in theconventional common culture methods such as productivity.

Although the fermentation broth obtained by the method of the presentinvention contains carbonate ions and/or bicarbonate ions, these areeasily emitted into air by heating, and therefore a fermentation brothor fermentation product with a large amount of the basic substancepresent as a solid can be obtained. Furthermore, when purification isneeded, if an acid stronger than carbonic acid is added to thefermentation broth, carbonate can be easily replaced with the strongeracid without performing ion exchange, which is usually performed in theconventional production methods. Furthermore, crystals of lysinehydrochloride can be directly obtained by concentrating the fermentationbroth.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments is incorporated by reference herein in its entirety.

What is claimed is:
 1. A method for producing a basic amino acid byfermentation comprising culturing a microorganism having an ability toproduce the basic amino acid in a liquid medium contained in afermentation tank to produce and accumulate the basic amino acid in themedium; wherein the amount of sulfate and/or chloride ions used ascounter ions of the basic amino acid is reduced by adjusting the totalammonia concentration in the medium to be within a specificconcentration range during at least a part of the total period of theculture process including a period where the pH of the medium increasesdue to shortage of the counter ions caused by accumulation of theobjective basic amino acid; wherein the total ammonia concentration inthe medium is adjusted so to ensure growth of the microorganism or theproduction of the basic amino acid, and at said concentration does notinhibit the production of the basic amino acid by the microorganism; andwherein the microorganism is an Escherichia coli bacterium or acoryneform bacterium.
 2. The method according to claim 1, wherein thetotal ammonia concentration in the medium is adjusted by adding ammoniaor urea to the medium when the activity of the microorganism is reducedor ceases as determined based on the indicators: dissolved oxygenconcentration in the medium, consumption rate of carbon source in themedium, turbidity of the medium, productivity of the basic amino acid,and pH change in the medium.
 3. The method according to claim 1, whereinthe medium comprises sulfate ions and/or chloride ions as a counter ionsource of the basic amino acid in an amount sufficient for performingthe culture at pH 7.2 or lower except that amount of sulfate ions and/orchloride ions is reduced by a desired amount, and the at least a part ofthe total period is a period where the pH of the medium cannot bemaintained to be 7.2 or lower due to a shortage of counter ions for thebasic amino acid which has accumulated in the medium.
 4. The methodaccording to claim 1, wherein the total ammonia concentration in themedium is adjusted to be 300 mM or lower.
 5. The method according toclaim 1, wherein the total ammonia concentration in the medium isadjusted to be 200 mM or lower.
 6. The method according to claim 1,wherein the total ammonia concentration in the medium is adjusted to be100 mM or lower.
 7. The method according to claim 1, which comprises astep of proliferating the microorganism.
 8. The method according toclaim 7, wherein the total ammonia concentration is not adjusted duringthe step of proliferating the microorganism.
 9. The method according toclaim 1, wherein the basic amino acid is selected from the groupconsisting of L-lysine, L-arginine and L-histidine.
 10. The methodaccording to claim 9, wherein the basic amino acid is L-lysine.
 11. Themethod according to claim 9, wherein the basic amino acid is L-arginine.12. The method according to claim 1, wherein the medium or a processedproduct thereof is heated after the culturing to eliminate bicarbonateions and carbonate ions.
 13. The method according to claim 1, whereinsaid method results in a product selected from the group consisting of afermentation broth, a dried product thereof, and products obtained byprocessing the fermentation broth or dried product thereof.
 14. Themethod according to claim 1, wherein the coryneform bacterium isCorynebacterium or Brevibacterium.